1. Field of the Invention
The present invention relates generally to the field of molecular biology, and relates to methods for amplifying nucleic acid target sequences in microfabricated devices. It particularly relates to isothermal methods for amplifying nucleic acid targets in microfabricated devices. The present invention also relates to methods of detecting and analyzing nucleic acids in microfabricated devices.
2. Description of Related Art
In vitro nucleic acid amplification techniques have provided powerful tools for detection and analysis of small amounts of nucleic acids. The extreme sensitivity of such methods has lead to their development in the fields of diagnosis of infectious and genetic diseases, isolation of genes for analysis, and detection of specific nucleic acids as in forensic medicine.
Nucleic acid amplification techniques may be grouped according to the temperature requirements of the procedure. Certain nucleic acid amplification methods, such as the polymerase chain reaction (PCR(trademark)xe2x80x94Saiki et al., 1985), ligase chain reaction (LCRxe2x80x94Wu et al., 1989; Barringer et al., 1990; Barony, 1991), transcription-based amplification (Kwoh et al., 1989) and restriction amplification (U.S. Pat. No. 5,102,784), require temperature cycling of the reaction between high denaturing temperatures and somewhat lower polymerization temperatures. In contrast, methods such as self-sustained sequence replication (3SR; Guatelli et al., 1990), the Qxcex2 replicase system (Lizardi et al., 1988), and Strand Displacement Amplification (SDAxe2x80x94Walker et al., 1992a, 1992b; U.S. Pat. No. 5,455,166) are isothermal reactions that are conducted at a constant temperature, which is typically much lower than the reaction temperatures of temperature cycling amplification methods.
The SDA reaction initially developed was conducted at a constant temperature between about 37xc2x0 C. and 42xc2x0 C. (U.S. Pat. No. 5,455,166). This was because the exo xe2x88x92klenow DNA polymerase and the restriction endonuclease (e.g., HindII) are mesophilic enzymes that are thermolabile (temperature sensitive) at temperatures above this range. The enzymes that drive the amplification are therefore inactivated as the reaction temperature is increased.
Methods for isothermal Strand Displacement Amplification, which may be performed in a higher temperature range than conventional SDA (about 50xc2x0 C. to 70xc2x0 C., xe2x80x9cthermophilic SDAxe2x80x9d), were later developed. Thermophilic SDA is described in European Patent Application No. 0 684 315 and employs thermophilic restriction endonucleases that nick the hemimodified restriction endonuclease recognition/cleavage site at high temperature and thermophilic polymerases that extend from the nick and displace the downstream strand in the same temperature range.
Photolithographic micromachining of silicon has been used to construct high-throughput integrated fluidic systems for a variety of chemical analyses. This technology is of particular interest for the development of devices for analysis of nucleic acids, as in their conventional formats such analyses are typically labor- and material-intensive. Ideally, all of the processing steps of the amplification reaction would be conducted on the microfabricated device to produce a completely integrated nucleic acid analysis system for liquid transfer, mixing, reaction and detection that requires minimal operator intervention.
Silicon and glass devices are economically attractive because the associated micromachining methods are, essentially, photographic reproduction techniques. Silicon structures are processed using batch fabrication and lithographic techniques. These processes resemble those of printing where many features may be printed at the same time. These processes permit the simultaneous fabrication of thousands of parts in parallel, thus reducing system costs enormously. Today, silicon fabrication techniques are available to simultaneously fabricate micrometer and submicrometer structures on large-area wafers (100 cm2), yielding millions of devices per wafer and may be used to process either silicon or glass substrates.
These characteristics have led to the proposal of silicon and glass as a candidate technology for the construction of high-throughput DNA analysis devices (Woolley and Mathies, 1994; Northrup et al., 1993; Effenhauser et al., 1994). As mechanical materials, both silicon and glass have well-known fabrication characteristics (Petersen, 1982). Microfabricated devices for biochemical and fluidic manipulation are undergoing development in many laboratories around the world (Ramsey et al., 1995; McIntyre 1996). Over the past 10 years, a number of microfluidic devices have been developed that allow the construction of miniaturized xe2x80x9cchemical reactors.xe2x80x9dIndividual components of the system such as pumps (Esashi et al., 1989; Zengerle et al., 1992; Matsumoto and Colgate, 1990; Folta et al., 1992); valves (Esashi et al., 1989, Ohnstein et al., 1990; Smits, 1990); fluid channels (Pfahler et al., 1990); chromatographic and liquid electrophoresis separation systems (Terry et al., 1979; Harrison et al., 1992b-g; Manz et al., 1991; Manz et al., 1992) are available. Although an objective of several research groups, complete silicon-fabricated nucleic acids analysis systems are still at the earliest stages of development.
Other components that have been microfabricated which are applicable to nucleic acid analysis include elements for gel electrophoresis (Zeineh and Zeineh, 1990; Heller and Tullis, 1992; Effenhauser et al., 1994; Woolley and Mathies, 1994, 1995; Webster et al., 1996); capillary electrophoresis (Manz et al. 1992, 1995; Effenhauser et al., 1993; Fan and Harrison, 1994; Jacobsen et al., 1994a; 1994b; Jacobson and Ramsey, 1995; Ocvirk et al., 1995; von Heeren et al., 1996); synthetic oligonucleotide arrays (Fodor et al., 1993; Schena et al., 1995; Hacia et al., 1996); continuous flow pumps (Lintel, 1988; Esashi et al., 1989; Matsumoto and Colgate, 1990; Nakagawa et al., 1990; Pfahler et al., 1990; Smits, 1990; Wilding et al, 1994; Olsson et al., 1995); discrete drop pumps (Burns et al., 1996); enzymatic reaction chambers (Northrup et al., 1994; Wilding et al., 1994b; Cheng et al., 1996); optical/radiation detectors (Belau et al., 1983; Wouters and van Sprakelaar, 1993; Webster et al., 1996); and multicomponent systems (Harrison et al., 1992, 1995; Northrup et al. 1994; Jacobsob and Ramsey 1996).
To date, a number of devices have been micromachined, including pumps and valves (Gravensen et al., 1993; Manz et al., 1994; Colgate and Matsumoto, 1990, Sammorco et al, 1996); reaction chambers (Woolley and Mathies, 1994; Wilding et al., 1994); and separation and detection systems (Weber and May, 1989, Northrup et al., 1993, Harrison et al., 1993; Manz et al., 1992; Jacobson et al., 1994; Schoonevald et al., 1991; Van den Berg and Bergveld, 1995; Woolley et al., 1995). Some of these have been recently integrated together to build pharmaceutical drug closing systems (Lammerink et al., 1993; Miyake et al., 1993) and other microchemical systems (Nakagawa et al., 1990; Washizu, 1992; Van den Berg and Bergveld, 1995). One device is an integrated glass system combining DNA restriction enzyme digestion and capillary electrophoresis (Jacobson and Ramsey 1996). An alternative format using high-density arrays of synthesized oligodeoxynucleotides has been demonstrated as a DNA sequence detector (Fodor et al., 1993; Hacia et al., 1996).
Nucleic acid targets have been successfully amplified by the PCR(trademark) on such microfabricated devices, often referred to as xe2x80x9cchipsxe2x80x9d (U.S. Pat. No. 5,498,392; Woolley et al., 1996; Shoffner et al., 1995; Cheng et al., 1996; Wilding et al., 1994; U.S. Pat. No. 5,589,136; U.S. Pat. No. 5, 639,423; U.S. Pat. No. 5,587,128, U.S. Pat. No. 5,451,500) and LCR (Cheng et al., 1996; U.S. Pat. No. 5,589,136). Evaporation due to repeated exposure to high temperatures during thermocycling is a problem. Evaporation during PCR(trademark) has been controlled by immersing the channel in oil such that the open ends are covered, but this makes recovery of the amplified sample difficult.
The present invention overcomes the foregoing evaporation and recovery drawbacks, and other deficiencies inherent in the prior art, by providing compositions and methods for use in the isothermal amplification of nucleic acids in microfabricated devices. In contrast to the difficulties previously perceived to exist and the prejudices in the art, the inventors found isothermal amplification of nucleic acids using microfabricated devices or xe2x80x9cchipsxe2x80x9d to be surprisingly effective. In fact, the chip-based isothermal amplification of the present invention was discovered to be efficient at previously untested low temperatures, despite potentially negative effects of surface chemistry and other proposed problems, such as stagnant temperature gradients, reduced diffusion and mixing, and inhibition of enzyme activity.
The invention thus generally provides an apparatus, system, device or chip, or a plurality thereof, with isothermally regulated reaction chambers, methods of constructing single-chip and multiple-chip analytical systems, and methods for using such devices, chips and systems in the isothermal amplification of nucleic acids. The invention also provides for the analysis of the amplification products using, e.g., sequencing, gel separation, and/or detection of the amplification products in microfabricated devices. Further methods of the invention therefore include laboratory methods connected with nucleic acid analysis and clinical methods connected with the diagnosis and prognosis of disease states.
First provided by the invention are devices, chips, wafers or an analytical apparatus or system(s), generally of a microfabricated or micromachined type, for use in the isothermal amplification of selected nucleic acids. Certain preferred devices utilize the silicon chip or silicon wafer formats. In preferred embodiments, the devices of the present invention are xe2x80x9cmicrodevicesxe2x80x9d, preferably defining micromachined structures for use with nanoliter volumes.
The apparati, devices or chips of the invention generally comprise a microfabricated substrate or housing defining at least a first transport channel, or microdroplet transport channel, operably connected to at least a first reaction chamber, and at least a first means for isothermally regulating the temperature of the reaction chamber.
The xe2x80x9cmeans for isothermally regulating the temperature of the reaction chamberxe2x80x9d may be an element, such as a particular resistor, combination of resistors, feed-back temperature detector, and/or circuitry for temperature control, that has not been previously used in conjunction with a microfabricated device or chip for use in nucleic acid amplification. More preferably, the xe2x80x9cmeansxe2x80x9d for isothermally regulating the temperature of the reaction chamber will be a xe2x80x9cprogrammable meansxe2x80x9d. That is, a series of executable and controlled steps, preferably in the form of a computer program, the implementation of which results in the control of the temperature of the reaction chamber within narrow limits, such that the temperature is xe2x80x9csubstantially constantxe2x80x9d. These computer microprocessor or programmable means, although readily prepared by those of skill in the art, have not previously been proposed for use in combination with a microfabricated nucleic acid amplification device.
The microfabricated substrate of the device, chip or system is generally constructed so that application of a fluid in one or more transport channels will result in the fluid being conveyed at least to the reaction chamber. Accordingly, the microfabricated substrate inherently has a xe2x80x9cflow-directed fabricationxe2x80x9d. The flow-directed fabrication or construction may be based upon gravitational attraction, thermal gradients, gas or liquid pressure differences, differences in hydrophobic and hydrophilic surface structures, electrowetting, and/or differences in the dielectric constant between reagent fluids applied to the substrate and the air or surrounding media. The manner in which a directional flow capability is provided to the substrate is not critical to the invention, so long as the substrate, device or system ultimately allows for the controlled manipulation of liquids or fluids applied thereto, and effective merging and mixing where appropriate.
In the context of this invention, a xe2x80x9creactionxe2x80x9d or xe2x80x9camplificationxe2x80x9d generally refers to reactions involving nucleic acid biomolecules, such as RNA and DNA. xe2x80x9cNucleic acid amplificationxe2x80x9d generally refers to the process of increasing the concentration of nucleic acid, and in particular, the concentration of a selected nucleic acid and/or a defined piece of a selected nucleic acid. xe2x80x9cAmplified or amplification productsxe2x80x9d or xe2x80x9campliconsxe2x80x9d generally define the products resulting from execution of a nucleic acid amplification reaction.
As used herein, the term xe2x80x9can isothermal amplification reactionxe2x80x9d refers to a nucleic acid amplification reaction that is conducted at a substantially constant temperature. It will be understood that this definition by no means excludes certain, preferably small, variations in temperature but is rather used to differentiate the isothermal amplification techniques from other amplification techniques known in the art that basically rely on xe2x80x9ccycling temperaturesxe2x80x9d in order to generate the amplified products. Thus, the present invention is distinguished from PCR, which fundamentally rests on the temperature cycling phenomenon.
It will be further understood that although the isothermal amplification reactions of the present invention will generally be conducted at a substantially constant temperature, the overall execution of the amplification, diagnostic or prognostic methods of the invention may nonetheless require certain steps to be conducted at different temperatures. For example, moving fluids or microdroplets through the different channels or chambers defined on the microfabricated substrate, and/or merging and mixing samples and reagents, may involve alterations in temperature, e.g., as may be achieved via the use of defined heating elements.
The microfabricated substrate or housing of the invention may be fabricated from any one of a number of suitable materials. The materials will preferably be of the type that can be manipulated to define the channels, reaction chambers and other components necessary for conducting the amplification methods, and yet will be stable enough to permit repeated use in such methods once the defining components have been etched or otherwise imparted onto the substrate. Certain preferred examples include, but are not limited to, silicon, quartz and glass.
The transport channels or xe2x80x9cmicrodroplet transport channelsxe2x80x9d defined in the substrate are generally pathways, whether straight, curved, single, multiple, in a network, etc., through which liquids, fluids and/or gases may be passively or actively transported. The channels are generally etched into the silicon, quartz, glass or other supporting substrate. The present invention requires the presence of at least a first channel that functions to allow the transport of a fluid sample into the reaction chamber. It will be understood that such a channel need not be of a significant minimum length, and that the term xe2x80x9cchannelxe2x80x9d therefore refers to a fluid-conveying section in functional terms, rather than to defining a structure that is necessarily long and pipe-like.
The one or more channels in the substrate connect the various components, i.e., keep components xe2x80x9cin communicationxe2x80x9d and more particularly, xe2x80x9cin fluidic communicationxe2x80x9d and still more particularly, xe2x80x9cin liquid communication.xe2x80x9d Such components include, but are not limited to, gas-intake channels and gas vents. In certain other aspects of the invention xe2x80x9cmicrodroplet transport channelsxe2x80x9d may refer to channels configured (in microns) so as to accommodate xe2x80x9cmicrodroplets.xe2x80x9d
While it is not intended that the present invention be limited by precise dimensions of the channels or precise volumes for microdroplets, illustrative ranges for channels may be between 0.5 and 50/xcexcm in depth (preferably between 5 and 20 xcexcm) and between 20 and 1000/xcexcm in width (preferably 500/xcexcm), and the volume of the microdroplets may range (calculated from their lengths) between approximately 0.01 and 100 nanoliters (more typically between ten and fifty).
The first microdroplet transport channel may be operably or functionally connected to, or in liquid communication with, at least a second microdroplet transport channel. First and second channels may operatively interact prior to connection with at least a first isothermally regulated reaction chamber. This would be the first meaning of xe2x80x9cconnective channelsxe2x80x9d. However, other operative connections are envisioned, and separate transport channels that function to deliver fluids to a common reaction chamber are still xe2x80x9cinteractive transport channelsxe2x80x9d in the context of the present invention in that they convey their contents to a common destination.
The present invention is not limited to the number of transport channels or other fluid-conveying means that may be provided in the substrate. The number and configuration of such channels will generally be dictated by the number of reaction chambers and other components provided on the substrate and/or the interaction of various individual chip elements to form a coordinated system.
At least one isothermally regulated reaction chamber is an important element of the present invention. As used herein, an xe2x80x9cisothermally regulated reaction chamberxe2x80x9d is a chamber, preferably one defining a microvolume receptacle, the temperature of which chamber may be regulated in order to keep it substantially constant. The xe2x80x9csubstantially constantxe2x80x9d temperature may be controlled within a few degrees, or within a single degree, or in certain embodiments, within a few tenths of a degree.
The means for isothermally regulating the reaction chamber may include, but are not limited to, resistors in contact with or in proximity to the reaction chamber, temperature detectors, resistive temperature detectors, dielectric sensors, or diodes and/or circuitry for temperature control. As discussed, the isothermal regulation means will preferably be a programmable means. The actual means of conveying the heat will preferably be a sheet resistively heated (rather than a wire), although polysilicon and doped polysilicon and diaphragm-type heaters may also be used in the reaction chamber.
In certain embodiments of the present invention, the microfabricated substrate further defines at least a first entry port operably or functionally connected to, or in liquid communication with, at least a first microdroplet transport channel. Any one of a variety of entry valves or ports may be used to control application of the sample or samples.
In embodiments where the microfabricated substrate further defines at least a second microdroplet transport channel, at least a second entry port may be provided in operable or functional connection, or in liquid communication with the second microdroplet transport channel. The invention is not limited to the number of transport channels, nor to the number of entry ports, either in terms of ports per channel or the total number of entry ports.
xe2x80x9cExit portsxe2x80x9d or xe2x80x9csample collection pointsxe2x80x9d are also envisioned, which are generally positioned at a downstream flow site from the reaction chamber.
In certain aspects of the invention, the microfabricated substrate will further comprise a flow-directing means system in order to facilitate that directed manipulation of fluids around the substrate. The term xe2x80x9cflow-directing means systemxe2x80x9d is intended to refer to one or more modifications of the substrate or other components used in functional association with the substrate that act to control, or further control, the transport, merging and/or mixing of fluids or microdroplets between the components etched onto the underlying substrate.
Certain preferred flow-directing means systems are those that employ a surface-tension-gradient mechanism in which discrete droplets are differentially heated and propelled through etched channels. A series of heating elements may thus be arrayed along the one or more microdroplet transport channels. Such resistive heaters may be located slightly beneath the channels. In certain aspects of the invention, the heating elements are comprised of aluminum, although one or more or a combination of other suitable resistive metals or materials may be employed, such as platinum, gold, etc.
In certain aspects of the invention, xe2x80x9cheating elementxe2x80x9d may refer to an element that is capable of at least partially liquefying a meltable material. A meltable material is xe2x80x9cassociated withxe2x80x9d a heating element when it is in proximity to the heating element such that the heating element can at least partially melt the meltable material. The proximity necessary will depend on the melting characteristics of the meltable material as well as the heating capacity of the heating element. The heating element may or may not be encompassed within the same substrate as the meltable material.
Other fluid-directing means systems for use in the invention are those that comprise a gas source in fluid communication with the one or more transport channels and other components, such that application of differential gas pressure gradients result in the controlled flow of gases or liquids through the micromachined device.
Differences in hydrophobic and hydrophilic surface structures may also be employed to control the flow or transport of fluids through the defined channels and etched components. In such embodiments, the transport channels and/or components may comprise or may be manipulated to comprise one or more hydrophobic regions. The channels and components may also be treated with a hydrophilicity-enhancing compound or compounds prior to addition of one or more of the biological samples or amplification reaction reagents.
xe2x80x9cHydrophilicity-enhancing compoundsxe2x80x9d are generally those compounds or preparations that enhance the hydrophilicity (water affinity) of a component, such as a transport channel. xe2x80x9cHydrophilicity-enhancing compoundxe2x80x9d is thus a functional term, rather than a structural definition. For example, Rain-X(trademark) anti-fog is a commercially available reagent containing glycols and siloxanes in ethyl alcohol. The fact that Rain-X(trademark) anti-fog renders a glass or silicon surface more hydrophilic is more important than the reagent""s particular formula.
In certain aspects of the invention xe2x80x9chydrophobic reagentsxe2x80x9d are used to make xe2x80x9chydrophobic coatingsxe2x80x9d and create xe2x80x9chydrophobic regionsxe2x80x9d (more water repellent) in channels. It will be understood that the present invention is not limited to particular hydrophobic reagents. In one embodiment, the present invention contemplates hydrophobic polymer molecules that may be grafted chemically to the silicon oxide surface. Such polymer molecules include, but are not limited to, polydimethylsiloxane. In another embodiment, the present invention contemplates the use of silanes to make hydrophobic coatings, including but not limited to halogenated silanes and alkylsilanes. The invention is not limited to particular silanes; the selection of the silane is only limited in a functional sense, i.e., that it render the surface hydrophobic.
In one embodiment, n-octadecyltrichlorosilane (OTS) is used as a hydrophobic reagent. In another embodiment, octadecyldimethylchlorosilane is employed. In yet another embodiment, the invention contemplates 1H, 1H, 2H, 2H-perfluorodecyltricholorosilane (FDTS, C10H4F17SiCl3) as a hydrophobic reagent. In still other embodiments, fluoroalkyl-, aminoalkyl-, phenyl-, vinyl-, bis silyl ethane- and 3-methacryloxypropyltrimethoxysilane (MAOP) are contemplated as hydrophobic reagents. Such reagents (or mixtures thereof) are useful for making hydrophobic coatings, and more preferably, useful for making regions of a channel hydrophobic (as distinct from coating the entire channel).
This invention is not limited to particular dimensions for the hydrophobic regions of the channels or components. However, while a variety of dimensions are possible, it is generally preferred that the regions have a width of between approximately 10 and 1000 xcexcm (or greater if desired), and more preferably between approximately 100 and 500 xcexcm.
A surface (such as a channel surface) is xe2x80x9chydrophobicxe2x80x9d when it displays advancing contact angles for water greater than approximately 70xc2x0. In one embodiment, the treated channel surfaces of the present invention display advancing contact angles for water between approximately 90xc2x0 and approximately 130xc2x0. In another embodiment, the treated microchannels have regions displaying advancing contact angles for water greater than approximately 130xc2x0.
In certain aspects of the invention, a xe2x80x9cliquid-abutting hydrophobic regionxe2x80x9d may refer to a hydrophobic region within a channel which has caused liquid (e.g., aqueous liquid) to stop or be blocked from further movement down the channel, said stopping or blocking being due to the hydrophobicity of the region, said stopped or blocked liquid positioned immediately adjacent to said hydrophobic region.
Other flow-controlling or flow-directing means systems contemplated for use in the present invention are those that rely on the phenomenon of electrowetting, and/or differences in the dielectric constant between the reagent fluids and air. Electrowetting may be described as the initial intake of fluid from a reservoir into a channel, electrowetting (or heating) may also be used to break the channel droplet from contact with the reservoir. Valve sealed by a movable diaphragm and/or meltable solder can also be used to control fluid flow.
Any one of a variety of pumps, both external and internal pumps, may be used in order to control the flow of fluids in the context of this invention. In certain aspects of the invention a xe2x80x9cbubble pumpxe2x80x9d may be used as a flow-directing means. A bubble pump operates as follows: fluid is introduced into a channel that comprises one or more electrodes positioned such that they will be in contact with a liquid sample placed in the channel. Two electrodes may be employed and a potential may be applied between the two electrodes. At both ends of the electrodes, hydrolysis takes place and a bubble is generated. The gas bubble continues to grow as the electrodes continue pumping electrical charges to the fluid. The expanded bubble creates a pressure differential between the two sides of the liquid drop which eventually is large enough to push the liquid forward and move it through the polymer channel.
When coupled with a capillary valve, a bubble pump can actuate an effective quantity of fluidic samples along the channel. The capillary valve is a narrow section of a channel. In operation, the fluidic sample is first injected into an inlet reservoir. As soon as the fluid is loaded, it moves in the channel by capillary force. The fluid then passes the narrow section of the channel but stops at the edge where the channel widens again. After the fluidic sample is loaded, a potential is applied between two electrodes. At both ends of the electrodes, hydrolysis occurs and bubble is generated. The bubble keeps growing as the electrodes continue pumping electrical charges to the fluid. The expanding bubble then creates a pressure differential between the two sides of the liquid drop, which eventually large enough to push the liquid forward.
The combination of bubble pump and capillary valve does not require any moving parts and is easy to fabricate. In addition, the device produces a well-controlled fluid motion, which depends on the bubble pressure. The bubble pressure is controlled by the amount of charges pumped by the electrodes. The power consumption of the device is also minimized by this method.
In certain aspects of the invention, the flow-directing means is separated from at least the first microdroplet transport channel by a liquid barrier. xe2x80x9cLiquid barrierxe2x80x9d or xe2x80x9cmoisture barrierxe2x80x9d refers to any structure or treatment process on existing structures that prevents short circuits and/or damage to electronic elements (e.g., prevents the destruction of the aluminum heating elements). In one embodiment, the liquid barrier may comprise a first silicon oxide layer, a silicon nitride layer and a second silicon oxide layer.
Further preferred aspects of the invention are those wherein the microfabricated substrate further defines, or is operably associated with, a nucleic acid analysis component operably connected to or in liquid communication with the isothermally regulated reaction chamber. The operative connection between the nucleic acid analysis component and the reaction chamber is such that the amplified nucleic acid products generated by the isothermal amplification reaction can be analyzed by the nucleic acid analysis component. The overall analytical method thus requires that the amplified products are conveyed or otherwise transported from the isothermally regulated reaction chamber to the nucleic acid analysis component in a manner effective to allow their subsequent analysis, separation, detection, or such like.
Any one of a variety of nucleic acid separation and analytical components may be used as part of the devices or systems of the present invention. Amplification product separation means include those for use in separation methods based upon chromatographic separation, including adsorption, partition, ion-exchange and molecular sieve, and techniques using column, paper, thin-layer and gas chromatography. Gel electrophoresis, liquid capillary electrophoresis, e.g., in glass, fused silica, coated and rectangular column format, polyacrylamide gel-filled capillary columns are particularly contemplated. Gel electrophoresis channels and/or capillary gel electrophoresis channels may thus be etched into the substrate.
The use of a miniature electrophoresis stage for macromolecule DNA separation is also contemplated. Using such a system can accomplish large savings of time and fluids by a reduction in sample size, an increase in processing system speed of the system, a increase in the number of samples handled through massive parallelism and batch fabrication techniques.
In certain embodiments, the present invention will comprises a nucleic acid detection means operably connected to, or in electrical communication with, the nucleic acid analysis component. Visualization means particularly envisioned include those using ethidium bromide/UV and radio or fluorometrically-labeled nucleotides, including antibody and biotin bound probes. The nucleic acid detection means may thus include, but is not limited to, a diode detection device with suitable filters for detection of radioactive decay, fluorescence, visible and nonvisible light wavelengths, and/or electromagnetic field changes.
The nucleic acid detection means may be a DNA sensor means, e.g., one that detects a radiolabel or a fluorescent label. Such DNA sensor means may be p-n-type diffusion diodes or p-n-type diffusion diodes combined with a wavelength filter and an excitation source. Silicon radiation/fluorescence detectors, photodiodes, silicon diffused diode detectors, and other silicon fabricated radiation detectors are also contemplated.
The control circuitry for preferable use in the device may be xe2x80x9con wafer control circuitryxe2x80x9d or xe2x80x9coff wafer control circuitryxe2x80x9d, the latter preferably for use in non-glass devices. In addition to the isothermal temperature controls, the control circuitry employed may include sample size and flow control circuits; timing circuits; electrophoretic separation bias, data detection and transmission control circuits; and one or more sequencer/timers to control the overall operation.
Thus the instant devices are contemplated for use in conducting a diagnostic test on a nucleic acid sample. Additionally, the present devices are contemplated for use in conducting a diagnostic or prognostic test on a biological sample suspected of containing a selected nucleic acid. Therefore, the present invention provides for the use of the instant devices in the manufacture of a kit or system for the amplification of nucleic acids. In certain aspects, the invention provides for the use of the present devices in the manufacture of a kit or system for the diagnosis or prognosis of a disease.
Any one or more of the isothermal amplification devices or chips of the present invention may be formulated or packaged with biological reagents effective to permit an isothermal nucleic acid amplification reaction. In such aspects, the combined reagents and devices may be considered as xe2x80x9cisothermal nucleic acid amplification kitsxe2x80x9d. xe2x80x9cBiological reagents effective to permit an isothermal nucleic acid amplification reactionxe2x80x9d are exemplified by polymerases, nucleotides, buffers, solvents, nucleases, endonucleases, primers, target nucleic acids including DNA and/or RNA, salts, and other suitable chemical or biological components.
The kits may thus be defined as comprising, in suitable container means at least a first microfabricated substrate defining at least a first channel, the at least a first channel connected to an isothermally regulated reaction chamber, and reagents effective to permit an isothermal amplification reaction.
In such kits, the first microfabricated substrate may further define a nucleic acid analysis component operably connected to said isothermally regulated reaction chamber and, optionally, a nucleic acid detection means operably connected to the nucleic acid analysis component.
The biological reagents effective for use in the amplification reactions may be provided or packaged in any suitable form, preferably aliquoted into suitable quantities. In certain preferred aspects, such reagents will be provided in a dry or lyophilized formulation. The provision of reagents, preferably in a lyophilized form, applies to both kits, in which the reagents are generally separately packaged, and integral devices, in which the lyophilized reagents may be pre-fabricated into one or more etched components on the substrate.
In certain other embodiments, an effective amount of the amplifying reagents may be provided in a separate cartridge that is interchangeably connected to the device, chip or system. Such replaceable cartridges or reservoirs may be provided in the same overall container means as the device, chip or system or may be purchased separately as distinct items. Different replaceable cartridges may be provided for conducting the various different isothermal amplification reactions that are known in the art. A number of reagent formulations may be packaged together for alternative use according to the needs of the end user.
Diagnostic systems are also provided by the present invention, comprising at least a first microfabricated substrate defining at least a first channel that is connected to at least a first isothermally regulated reaction chamber; wherein the diagnostic system further comprises a nucleic acid analysis component and a nucleic acid detection means in operable association with the reaction chamber of the microfabricated substrate.
The diagnostic systems may also comprise, in operable association, at least a second microfabricated substrate defining at least a second channel that is connected to at least a second isothermally regulated reaction chamber. Third, fourth, fifth, tenth, 20th, 50th, 100th, 500th and 1000th microfabricated substrates may also be provided, as is the meaning of xe2x80x9ca plurality of microfabricated substratesxe2x80x9d.
The diagnostic systems may variously have at least a first and at least a second microfabricated substrate, or a plurality thereof, that are operably connected in series to a single nucleic acid analysis component and nucleic acid detection means. The diagnostic systems may alternatively comprise at least a first and at least a second microfabricated substrate, or a plurality thereof, that are operably connected in parallel to at least two distinct nucleic acid analysis components and nucleic acid detection means, or a plurality of such components.
In such kit and system embodiments, liquid handling, electrophoresis, and detector components may be coupled into an integrated format. DNA samples may move directly between sample processing, size-separation, and product detection. The components are controlled by electronic circuitry, fabricated on the same silicon wafer.
Accordingly, an integrated DNA sample processing design may be arrayed as multiple parallel units on a single silicon wafer. The number of parallel DNA processing units per wafer may be maximized, and circuitry used for overall control. A large number of simultaneous isothermal amplification reactions (up to 1000 per wafer) may be performed on such systems.
Methods of making devices for use in isothermal nucleic acid amplification are provided by the invention, which generally comprise preparing at least a first microfabricated device, chip or wafer defining at least a first channel that is operably connected to an isothermally regulated reaction chamber, preferably isothermally regulated by a programmable means.
A method of making a nucleic acid diagnostic kit is also provided, which generally comprises preparing at least a first microfabricated device, chip or wafer defining at least a first channel that is operably connected to an isothermally regulated reaction chamber, and combining the microfabricated device with biological reagents effective for use in an isothermal amplification reaction. The combination may be with lyophilized reagents, which may further be disposed in the device as an integral component.
Methods of making a nucleic acid diagnostic system are further provided, comprising preparing at least a first microfabricated substrate defining, in a series of operable associations, at least a first channel, an isothermally regulated reaction chamber, a nucleic acid analysis component and a nucleic acid analysis detection means.
Multi-component nucleic acid diagnostic systems may also be manufactured by the methods of the present invention. To make a multi-component nucleic acid diagnostic system, a plurality of microfabricated substrates, nucleic acid analysis and detection means are operably combined, preferably in an interactive array or arrays. Controlling electronic circuitry and programmable regulating means are preferably provided. Multiple parallel unit arrays on single silicon wafers are particularly preferred.
Important aspects of the present invention are methods for the isothermal amplification of selected nucleic acids or portions thereof, which methods generally comprise providing or introducing a microdroplet sample comprising or suspected of comprising the selected nucleic acid, and reagents effective to permit an isothermal amplification reaction, to at least a first microfabricated substrate with an isothermally regulated reaction chamber, as generally defined hereinabove, and conducting an isothermally regulated amplification reaction to amplify the selected nucleic acid or a portion thereof.
As used herein, the terms xe2x80x9cprovidingxe2x80x9d or xe2x80x9cintroducingxe2x80x9d mean that the sample or samples are provided or introduced into the one or more microfabricated substrates in a manner effective to begin their conveyance, transportation or general movement to the isothermally regulated reaction chamber. As described hereinabove, a number of particular flow-directing means systems may be employed in order to convey the sample or samples to the reaction chamber. Where differential heating is employed as the sole transport means, or as part of the overall transport means, an important aspect of the invention is that any samples that comprise enzymes for use in the isothermally regulated nucleic acid amplification reaction are xe2x80x9cthermotransportedxe2x80x9d at a temperature below the critical temperature of the polymerase enzyme. Preferably, all samples will be transported at temperatures that are below the critical ranges for substantial inactivation of the enzymes for use in the isothermal amplification reaction. It is a surprising feature of the invention that heat-conveying temperatures effective to transport samples into the reaction chamber can be employed that are far enough below the denaturation and/or inactivation temperatures of the enzymes necessary to catalyze the isothermal nucleic acid amplifications. The invention may thus be characterized as including a method step of conveying said sample and/or said reagents from an initial contact point on the microfabricated substrate to the isothermally regulated reaction chamber at a xe2x80x9ctransportingly effective temperaturexe2x80x9d that does not significantly denature the selected amplification enzyme or otherwise significantly impair or reduce its catalytic amplification activity.
The isothermal amplification reactions of the invention are also conducted at temperatures effective and by means effective to result in productive mixing of the one or more samples and amplification reagents. xe2x80x9cEffective mixingxe2x80x9d is a functional term, most readily characterized by the operative execution of the amplification reaction such that amplified products may be detected. If desired, one or more samples containing nucleic acids and/or amplification reagents may first be xe2x80x9cmergedxe2x80x9d prior to mixing.
In certain definitional terms, xe2x80x9cmergingxe2x80x9d is distinct from xe2x80x9cmixing.xe2x80x9d When a first and second microdroplet is merged to create a merged microdroplet, the liquid may or may not be mixed.
In any event, irrespective of the degree of prior sample association, the isothermal amplification reaction as a whole must be conducted under conditions effective to adequately mix the substrates and other components of the reaction. Prior to the present invention, it was generally believed in the art that effective mixing could not be achieved at the temperatures preferred for use in the present isothermal amplification reactions. Only the endeavors of the present inventors, conducted despite the prejudices in the prior art, resulted in the discovery that effective mixing could be achieved. Effective mixing is achievable despite the viscosity of the samples and/or reagent formulations used, and the particular biological components employed in connection with the isothermal amplification enzyme solutions and/or suspensions.
Those of ordinary skill in the art will be able to vary the application of the samples and reagents and the manner of transporting such components to the reaction chamber, in addition to varying the particular details of the amplification reaction, in order to ensure that a degree of mixing sufficient to result in amplified products is achieved. Moreover, the degree of mixing in a merged microdroplet may be enhanced by a variety of techniques provided by the present invention, including but not limited to, reversing the flow direction of the merged microdroplet (as discussed herein below).
Although not in any way being limited by the following guidance, the temperature differential believed to be effective in conveying microdroplet samples along a microfabricated device in accordance with the present invention should generally be a temperature differential of at least about 10xc2x0 C. Preferably, temperature differentials of at least about 11xc2x0 C., 12xc2x0 C., 13xc2x0 C., 14xc2x0 C., 15xc2x0 C., 16xc2x0 C., 17xc2x0 C., 18xc2x0 C., 19xc2x0 C., 20xc2x0 C., about 25xc2x0 C., about 30xc2x0 C., about 35xc2x0 C. or even up to about 40xc2x0 C. or above may be advantageously used in conveying microdroplet samples along a micromachined device or substrate. It will be understood that each of the foregoing effective conveying temperature differentials must be analyzed in connection with the preferred operating temperatures for any one or more particular amplifying enzyme, and that the temperatures chosen must be below the temperature at which the enzyme denatures or otherwise becomes significantly impaired in its catalytic activity. In general, it is believed that temperature differences of greater than about 30xc2x0 C. will be preferred for creating microdroplet motion or movement. In certain other embodiments, temperature differentials of about 40xc2x0 C. will be effective, and these temperature gradients can be readily generated by a number of means, particularly by the use of a series of temperature sensors arrayed along the entire length of the one or more conveying channels etched into the substrate.
Although an understanding of the mechanisms of action underlying the surprising operability of the present invention is not necessary in order to carry out the claimed amplification methods, the inventors further point out that circulation patterns generated in the drop during motion aid in mixing the liquid sample. Studies using metal elements as both heaters and temperature sensors demonstrate that a temperature differential of 20-40xc2x0 C. across the drop is sufficient to provide forward motion in transport channels.
Thus for only small temperature differences across the drop (on the order of 10xc2x0 C.) velocities on the order of 1 cm/s may be obtained. This velocity is more than sufficient for transporting liquid drops in MIDAT and other chip based systems.
Those of ordinary skill in the art will further understand that other physical components of the chip fabrication will impact the temperatures effective to transport microdroplets. By way of example only, in studies using glass capillaries, it has been found that there is a minimum temperature difference required to move the droplet. For instance, if the advancing angle is 36xc2x0 and the receding angle is 29xc2x0 (with the front of the droplet being 25xc2x0 C.), then the back of the droplet would need to be heated to xcx9c60xc2x0 C. for a 1 mm long droplet in a 20 mm high channel. This is just one example situation.
The use of channel geometry and defined chip fabrications that necessitate higher transport temperatures will naturally be combined with the use of enzymes that are functional at higher isothermal amplification temperatures. The choice of enzyme and transport temperatures will be routine to those of ordinary skill in the art, with a number of possibilities being readily available. By way of example only, methods for isothermal SDA are available in which temperatures of between about 50xc2x0 C. and about 70xc2x0 C. are used in conjunction with a thermophilic amplification enzyme. Accordingly, temperatures of about 30xc2x0 C., about 35xc2x0 C., about 40xc2x0 C., about 45xc2x0 C., about 50xc2x0 C., about 55xc2x0 C., about 60xc2x0 C., about 65xc2x0 C., about 70xc2x0 C., also may be employed.
However, the calculations of the present inventors indicated that about a 35xc2x0 C. difference between the front and back of a droplet will be sufficient to initiate droplet motion in a system with advancing angles of 36xc2x0 and receding angles of 29xc2x0 in a 20 mm high channel. Further studies of effective transport showed that the resulting temperature difference was xe2x88x9240xc2x0 C. between the front and back of the droplet, thus corroborating the initial determination of the requirements.
This shows that the range of transporting temperatures and the variety of enzymes for use in the invention extends to encompass each of the enzymes known to be suitable for use in isothermal amplifications. For example, 3SR and Qbeta-replicase are known to function at 37xc2x0 C., which can be used as part of the effective conveying temperature. Classical SDA reactions can also be conducted at a constant temperature between about 37xc2x0 C. and 42xc2x0 C., the preferred range identified in U.S. Pat. No. 5,455,166 (incorporated herein by reference).
U.S. Pat. No. 5,455,166 is also incorporated herein by reference for the purposes of exemplifying the level of skill in the art regarding the selection of each component necessary for the isothermal amplification reaction. For example, this patent explains that, in addition to the DNA polymerases, the restriction endonucleases necessary to carry out the reaction are also mesophilic enzymes that are thermolabile at temperatures generally above the 37-42xc2x0 C. advised for use in the reaction. All such considerations will be readily employed by those of skill in the art as they select the reagents necessary for use in the present isothermal amplification reactions.
In terms of the isothermal amplification reaction itself, rather than the transporting, merging and/or mixing steps, those of ordinary skill in the art will instantly appreciate appropriate temperatures for use in connection with the selected polymerase, replicase or other amplification system. By way of example only, isothermal amplification reactions involving 3SR and Qbeta-replicase may be conducted at or about 37xc2x0 C. Standard SDA isothermal amplification reactions may be conducted at a constant temperature between about 37xc2x0 C. and 42xc2x0 C. (including 38xc2x0 C., 39xc2x0 C., 40xc2x0 C. and 41xc2x0 C.), whereas isothermal SDA using a the enzyme may be performed at a higher temperature range than conventional SDA, anywhere between about 50xc2x0 C. and about 70xc2x0 C.
Any effective temperature that will support the desired enzymatic activity, even if sub-optimal, may be employed in the isothermal amplification reactions of this invention. Accordingly, the isothermal amplifications may be conducted at any substantially constant and effective temperature, including at about 20xc2x0 C., 21xc2x0 C., 22xc2x0 C., 23xc2x0 C., 24xc2x0 C., 25xc2x0 C., 26xc2x0 C., 27xc2x0 C., 28xc2x0 C., 29xc2x0 C., 30xc2x0 C., 31xc2x0 C., 32xc2x0 C., 33xc2x0 C., 34xc2x0 C., 35xc2x0 C., 36xc2x0 C., 37xc2x0 C., 38xc2x0 C., 39xc2x0 C., 40xc2x0 C., 41xc2x0 C., 42xc2x0 C.,43xc2x0 C., 44xc2x0 C., 45xc2x0 C., 46xc2x0 C., 47xc2x0 C., 48xc2x0 C., 49xc2x0 C., 50xc2x0 C., 51xc2x0 C., 52xc2x0 C., 53xc2x0 C., 54xc2x0 C., 55xc2x0 C., 56xc2x0 C., 57xc2x0 C., 58xc2x0 C., 59xc2x0 C., 60xc2x0 C., 61xc2x0 C., 62xc2x0 C., 63xc2x0 C., 64xc2x0 C., 65xc2x0 C., 66xc2x0 C., 67xc2x0 C., 68xc2x0 C., 69xc2x0 C., 69xc2x0 C., 70xc2x0 C., 71xc2x0 C., 72xc2x0 C., 73xc2x0 C., 74xc2x0 C., 75xc2x0 C., and the like.
It will be understood that the overall isothermal amplification reaction is carried out in a manner effective to result in at least detectable amounts of amplified products. xe2x80x9cAt least detectable amounts of amplified productsxe2x80x9d refers to a yield of amplified nucleic acid products that can be detected by currently available nucleic acid detection means. Optical methods using efficient fluorophores can detect atto-molar concentrations (corresponding to xcx9c105 DNA molecules) migrating in capillary channels of 8xc3x9750 mm internal cross section (Woolley and Mathies, 1994; incorporated herein by reference). Reactions for synthesizing such DNA quantities can reasonably occur in 10 xcexcl. An integrated system designed for picoliter volumes may require channel dimensions on the order of 10 xcexcm2xc3x97100 xcexcm (cross sectionxc3x97length).
In contrast to the negative beliefs in the prior art, the present invention has provided methods for target amplification efficiency surprisingly equivalent to conventional SDA reactions, but conducted on a DNA chip. Amplifications of almost a million-fold have already been achieved. This demonstrated that the physical changes in the environment on the DNA chip, including silicon contact, temperature gradients, surface interactions and other potential inhibitors, did not adversely affect the amplification reaction.
In certain preferred embodiments, it is believed that the isothermal amplification reactions of the present invention will be conducted such that the sample nucleic acids are amplified at least about 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, 2000-fold, 5000-fold, 10,000-fold, 50,000-fold, 100,000-fold, 200,000-fold, 300,000-fold, 400,000-fold, 500,000-fold, 600,000-fold, 700,000-fold, 800,000-fold, 900,000-fold, or so, up to and including at least about 1,000,000-fold, 2,000,000-fold or so.
The simplicity of sample provision to microfabricated devices is another surprising feature of the present isothermal amplification methods. The samples may be provided in any xe2x80x9csilicon-compatible formulationxe2x80x9d. Prior to the present invention, it was not known whether the various isothermal polymerases and replicases would be operative in contact with the fabricating structures of a microdevice, particularly the preferred silicon formulations. The diligent studies of the present inventors have shown that the present isothermal amplification methods function in a xe2x80x9csilicon-compatible mannerxe2x80x9d, and the methods of the invention are intended to be carried out in such effective manners.
The provision of the sample to the microfabricated or micromachined devices or systems is not believed to be critical, so long as the samples are later capable of being conveyed along the appropriate channels. Sample sources include, but are not limited to, continuous streams of liquid as well as static sources (such as liquid in a reservoir). In a preferred embodiment, the source of liquid microdroplets comprises liquid in a microchannel from which microdroplets of a discrete size are split off. As described above, in certain preferred embodiments, the reagents for use in the isothermal reaction will already be comprised within a pre-fabricated microdevice. In such embodiments, lyophilized reagents may be rendered active by contact with the nucleic acid-containing sample, or alternatively, they may be separately contacted with another fluid sample, such as a buffer.
The samples comprising the nucleic acids for application in the present isothermal amplification methods may be xe2x80x9claboratory samplesxe2x80x9d for use in any one of a variety of molecular biological embodiments. Such samples may also be xe2x80x9cbiological or clinical samplesxe2x80x9d, in which case the samples will generally be obtained from or otherwise derived from an animal or human subject.
In any event, where the samples used are xe2x80x9cmicrodroplet samplesxe2x80x9d, this term generally refers to the microdroplet themselves and samples from which microdroplets may be made.
Whether the sample is a laboratory, biological or clinical sample, the purity of the nucleic acids within the sample may vary widely. The purity of the sample is controlled only by the need to have a minimum purity necessary for successful execution of the isothermal amplification reaction. In certain embodiments, the sample will have been subjected to a substantial degree of extraction or purification prior to use in the present invention, although this is not necessary in all embodiments.
In terms of the biological samples, these may be obtained from a variety of biological fluids, including blood, plasma, urine, sputum, semen, and fluids obtained from homogenized tissues. It is not believed to be necessary to limit the presence of other biological components, such as proteins and lipids, from the samples for use in the invention, although this may be desired in certain embodiments and is within the level of skill of the ordinary artisan.
In common with the sample preparation, the purity of the reactants provided to the device and the makeup of the device itself require some degree of biocompatability in order to achieve the desired reaction. That is to say, that the isothermal amplification reaction should not be substantially inhibited or prevented by any components present within the biological sample, contaminants within the reactants or by the characteristics or nature of the device components, including the silicon fabricants.
It will be understood that the particular components, amounts of components and/or reactants and the particular conditions of the reaction may be modified in order to optimize the isothermal amplification reaction itself. All such variations and modifications are routinely investigated in this field of study. By way of example only, one may vary the concentration of any of the components or the samples, the temperature, pH or ionic makeup of the buffers, and generally vary any other parameter of the amplification reaction.
It will be understood that the execution of the amplification reaction, including the application of the samples and the movement, mixing and distribution of the samples prior to the actual isothermal amplification step, may also require certain optimizations. All such variations and optimizations will be routine to those skilled in this field of study.
All liquid distributions and manipulations may be performed entirely within a handling system formed as channels in micromachined silicon. Sensors may monitor the temperature and location of liquid in the channels. The manipulation of reagents includes the movement, merging, mixture, and temperature control of the reagents to allow nucleic acid amplification under isothermal reaction conditions.
In certain aspects of the present invention, the isothermal amplification methods and the reagents provided for use in the methods will be based upon the strand displacement amplification reaction. Self-sustained sequence replication amplification reactions and/or Qxcex2 replicase amplification reactions may also be used.
A preferred technique is the Strand Displacement Amplification (SDA). The SDA reaction may be conducted at a substantially constant temperature between about 37xc2x0 C. and about 42xc2x0 C., or at any other effective temperature, as exemplified herein by 52xc2x0 C. It was previously believed that the low temperature requirement for SDA would prevent its use in connection with amplification on microchip devices. However, the inventors discovered that the potential problems of stagnant temperature gradients and reduced diffusion and sample mixing do not actually impact the efficiency of the SDA reaction in such microvolume embodiments.
Thermophilic SDA may also be employed, as described in published European Patent Application No. 0 684 315 (incorporated herein by reference). This technique employs thermophilic restriction endonucleases which nick the hemimodified restriction endonuclease recognition/cleavage site at high temperature and thermophilic polymerases which extend from the nick and displacing the downstream strand in the same temperature range. At increased temperature, the amplification reaction has improved specificity and efficiency, reduced nonspecific background amplification, and potentially improved yields of amplification products.
In terms of amplified product analysis, DNA samples may be size-fractionated on an electrophoresis system built within or attached or connected to the silicon substrate. Electrophoresed DNA products may be visualized by radioactivity or fluorescence detectors fabricated directly in the silicon wafer.
In certain aspects of the invention, the amplified nucleic acid is detected by means of a detectable label incorporated into the amplified selected nucleic acid by the isothermal amplification reaction. In other aspects, it is detected by means of a labeled probe. The label may variously be a radioisotopic, enzymatic or fluorescent label.
The present invention further provides methods for detecting the presence of a selected nucleic acid, comprising introducing a sample suspected of containing the selected nucleic acid, and reagents effective to permit an isothermal amplification reaction, into a microfabricated substrate defining at least a first channel, the at least a first channel connected to an isothermally regulated reaction chamber, conducting an isothermally regulated amplification reaction to amplify the selected nucleic acid, and detecting the presence of the amplified selected nucleic acid, wherein the presence of the amplified selected nucleic acid confirms the presence of the selected nucleic acid in the sample.
The sample may be obtained or derived from an animal or patient having or suspected of having a disease. It will be understood that in certain aspects of the present diagnostic and/or prognostic methods, the presence of the ultimate amplified selected nucleic acid will be indicative of the disease state being analyzed. In alternative embodiments, it is the absence of amplified nucleic acid products that is indicative of a disease state. In either embodiment, the present invention is ideally suited for the amplification of nucleic acids of defined sequence, having a defined sequence element, or including a potential point mutation, as each of the foregoing variants may be distinguished by analyzing the amplified products resulting from execution of the presently claimed methods.